1. Field of the Invention
This invention relates to methods for using volume exclusion agents to enhance in situ hybridization rates between short oligonucleotide probes and their target polynucleotides where the cells containing the target polynucleotides are adhered onto a glass substrate. In one aspect, the invention specifically relates to the use of volume exclusion agents to facilitate assay and diagnostic procedures for the detection of a virus, such as human papillomavirus (HPV). In addition, diagnostic kits embracing the above methods are described herein.
2. Information Disclosure
In situ hybridization is a method of locating or detecting target polynucleotides within intact cells using labeled polynucleotide probes. Typically, the probes have sufficient sequence complementarity to bind to the target under preselected hybridization conditions. The presence and amount of bound probes is determined in accord with the type of label attached to the probes.
Much of the research related to hybridization between target and probe polynucleotides for assay and diagnostic purposes has been directed toward optimizing rates of hybridization. In situ hybridization is particularly problematic due to the inability of the probes to readily enter into the nucleus or cytoplasm in which their target polynucleotides are located. To solve this problem, researchers have attempted, inter alia, to reduce the size of the probe and to alter cell fixation procedures to facilitate entry of the probe into the cytoplasm or nucleus, see generally Singer, R. H., et al., "Optimization of In Situ Hybridization Using Isotopic and Non-Isotopic Detection Methods," Biotechniques 4(3):230-250, 1986, and Haase, A., et al., "Detection of Viral Nucleic Acids by In Situ Hybridization," Methods in Virology, Vol. VII, pp. 189-226, (1984).
U.S. Pat. No. 4,302,204 has disclosed that the presence of volume exclusion agents may increase hybridization rates. This effect has been reported to be due to the exclusion of the probe molecules from the volume occupied by the volume exclusion agent, which allegedly results in an effective increase in probe concentration. Amasino, R. M., "Acceleration of Nucleic Acid Rate by Polyethylene Glycol," Anal. Biochem., 152:304-307 (1986). It has been reported that the effect of dextran sulfate, the most commonly used exclusion agent, was most pronounced in mixed phase hybridizations where the probes exceeded 250 nucleotides. Further, it has been reported that as the probe size decreases, so would the enhancing effect of dextran sulfate on the rate of hybridization, with no effect observed for oligonucleotides of 14 bases. Meinkoth J. and Wahl J., "Hybridization of Nucleic Acids Immobilized on Solid Supports" (Review), Anal. Biochem., 138:267-284 at 268 (1984). The use of volume exclusion to enhance in situ hybridization has also been reported. It was reported that an average length of 400 nucleotides is optimal for hybridization in situ in the presence of dextran sulfate. Hasse, A., supra. at 205.
U.S. Pat. No. 4,689,294 discloses the use of polyacrylate salts and polymethylacrylate salts to enhance the rate of hybridization between two complementary polynucleotide segments. U.S. Pat. No. 4,689,294 is incorporated herein by reference.
The in situ localization of HPV DNA using long biotinylated probes in the presence of dextran sulfate has also been reported by Beckmann, P. M., et al.; "Detection and Localization of Human Papillomavirus DNA in Human Genital Condylomas by In Situ Hybridization with Biotinylated Probes," J. Med. Virol., 16:265-273 (1985); Milde K., Loning, T., "Detection of Papillomavirus DNA in Oral Papillomas and Carcinomas: Application of In Situ Hybridization with Biotinylated HPV 16 probes," J. Oral Pathol., 15:292-296 (1986); and McDougall, J. K., et al., "Methods for Diagnosing Papillomavirus Infection," in Papillomaviruses, Wiley, Chicester (CIBA Foundation Symposium 120), pp. 86-103 (1986).
In the field of nucleic acid hybridization, the need for rapid assay tests for the accurate and reproducible detection of nucleic acids has been a long standing problem. Any procedures that demonstrate a tendency to accelerate the typically multi-hour long processes are of value, especially for hybridization assays to be conducted by clinical laboratories.
A U.S. patent application was filed on Oct. 2, 1987, entitled "Human Papillomavirus Type Diagnosis With Nucelotide Probes, Ser. No. 103,979, and is incorporated herein by reference.